Affinity separation is separation achieved by employing the specific binding of one molecule by another. Bioaffinity separation is defined as an affinity separation in which one of the components involved in an affinity reaction is biologically active or is of biological interest. Bioaffinity separations generally involve at least one biomacromolecule, such as a protein or nucleic acid, as one of the components of the binding pair. Examples of such bioaffinity binding pairs include: antigen-antibody, substrate-enzyme, effector-enzyme, complementary nucleic acid strands, and others; the terms ligand and binder will be used to represent the two components in specific bioaffinity binding pairs.
Affinity separations are often considered to require the use of solid supports derivatized with a ligand or binder. Such solid supports should have the following properties: physical and chemical stability; chemical inertness; compatibility with a variety of biological samples; utility in batch and chromatographic applications; ability to provide for ready and secure attachment of ligands and binders to the surface; and allow simple efficient regeneration of the support.
Solid perfluorocarbon polymer supports have been found to be especially effective as affinity supports. Applicant's assignee's, E. I. du Pont de Nemours & Co. U.S. Pat. Nos. 4,885,250 and 4,954,444, issued Dec. 5, 1989 and Jan. 5, 1990, respectively, disclose perfluorocarbon polymer supports to which perfluorocarbon-substituted ligands or binders have been attached. Such supports can be prepared by batch and chromatographic processes.
Perfluorocarbon polymer carrier surfaces are hydrophobic and adsorption of perfluorocarbon-substituted ligands and binders to these surfaces is made difficult by the inability to wet these surfaces with aqueous solutions. Upon contacting a perfluorocarbon polymer carrier with an aqueous solution containing a perfluorocarbon-substituted ligand or binder, it is believed that a layer of air is formed between the carrier surface and the aqueous solution causing the carrier to clump and float at the surface of the aqueous solution. The ligand or binder contained in such solution is unable to contact uniformly and adsorb to the carrier surface. Preparation of supports using only aqueous solutions can result in non-uniform and unsecure adsorption of ligands and binders on the support surfaces.
Treating perfluorocarbon polymer surfaces with organic solvents before adsorbing ligands and binders to these surfaces has been described. Janata et al. (U.S. Pat. No. 3,966,580, issued Jun. 29, 1976) disclose a protein-immobilizing hydrophobic polymeric membrane prepared by swelling the membrane with an organic solvent containing an aliphatic compound having a reactive site, drying the membrane to remove the solvent, and immersing the membrane in a solution containing a compound having both a reactive site which recognizes the reactive site of the aliphatic compound and a protein-reactive site. The membrane is then ready to be reacted with the protein to be immobilized. Lower molecular weight organic solvents are preferred so they can be readily removed.
Fishman (U.S. Pat. No. 3,843,443, issued Oct. 22, 1974) discloses two processes for preparing a bound polypeptide material. A porous unsintered fluorocarbon polymer is first flooded with a water-miscible organic solvent. The flooded fluorocarbon polymer is then contacted with an aqueous solution of polypeptide polyelectrolyte for a length of time sufficient to replace a portion of the organic solvent with the polypeptide polyelectrolyte. Alternatively, the solvent-flooded fluorocarbon polymer can be flooded with water prior to contacting it with the aqueous polypeptide polyelectrolyte solution. Neither process is suitable for preparing perfluorocarbon polymer supports. Since perfluorocarbon-substituted ligands and binders can be denatured by solutions containing a high concentration of water-miscible organic solvent, contacting an aqueous solution of such ligands or binders with a solvent-flooded carrier as in the first process can denature the ligands or binders. It is believed that when preparing a support using the second process, the carrier would become unwetted after flooding it with water and would float to the water surface. Subsequently contacting the carrier with an aqueous solution of a perfluorocarbon-substituted ligand or binder could result in non-uniform and non-secure adsorption of such ligand or binder.
WO 8603-840-A, published Jul. 3, 1986, discloses a process for treating a synthetic polymer to make it receptive to adsorption of an immunoreagent by thoroughly rinsing the polymer surface with a water-miscible organic solvent and then thoroughly rinsing the polymer surface with water. The solvent is used to clean the polymer surface before contacting the surface with the immunoreagent. It is believed that this process is not suitable for preparation of perfluorocarbon polymer supports because once water is added to a perfluorocarbon polymer carrier, it will become unwetted and float to the water surface. Adsorption of a perfluorocarbon-substituted ligand or binder to the carrier surface could be non-uniform and unsecure.
Applicant's assignee's patents, U.S. Pat. No. 4,885,250 and U.S. Pat. No. 4,954,444 listed above, describe a procedure for preparing solid perfluorocarbon polymer carriers by first contacting the carriers with methanol followed by aqueous methanol, water, and buffer prior to contacting the carriers with aqueous solutions of perfluorocarbon-substituted ligands or binders. While such procedure results in secure binding of ligands or binders, handling of the carriers can be difficult. Upon contacting the carriers with water, they become unwetted and float to the water surface. Small quantities of the carriers can be rewetted by high speed centrifugation, but centrifugation cannot rewet large scale batch quantities of carriers.
There exists a need for a convenient process for preparing solid perfluorocarbon polymer supports permitting uniform and secure adsorption of perfluorocarbon-substituted ligands or binders.